PROJECT SUMMARY Immunoregulation, including suppression of autoimmunity, is a fundamental function of a healthy immune system. FOXP3 is a key transcription factor for a subset of CD4+ T regulatory cells (Tregs), which can be of thymic or peripheral origin. Mutations in FOXP3 cause the Immune-dysregulation Polyendocrinopathy- Enteropathy- X-linked (IPEX) syndrome, an early onset deadly X-linked immunodeficiency with severe multiple autoimmune manifestations, most notably in the skin, GI tract, and endocrine organs. Loss-of- function FOXP3 mutations result in a primary dysfunction of Tregs leading to chronic inflammation, lymphoproliferation and autoantibody production. Currently, the only cure for IPEX syndrome is allogeneic hematopoietic stem cell transplantation (allo-HSCT) from a matched donor. The therapeutic benefit of allo-HSCT demonstrates that replacing the immune system with wild-type FOXP3 cells can cure the disease. However, the limited matched donor availability and toxicities of allo-HSCT make this approach challenging. The goal of this proposal is to develop a CRISPR/Cas9 gene editing approach for site- specific restoration of wild-type FOXP3 gene expression. We will apply this method to both T cells and hematopoietic stem cells (HSPCs) carrying FOXP3 mutations to correct Treg functional defects and to reverse the IPEX phenotype in preclinical disease models. The CRISPR/Cas9 genome editing system is an efficient gene editing tool in various cell types. We can achieve very high efficiency of gene editing in both CD34+ HSPC and stimulated or unstimulated T cells using chemically modified CRISPR guide RNAs (sgRNAs). In the present study, we will apply this to edit the FOXP3 gene locus i n IPEX patient cells. As mutations that cause IPEX are distributed throughout the FOXP3 gene, our gene editing strategy involves insertion of a wild-type FOXP3 cDNA copy at the initiation codon so that c D N A expression is regulated by endogenous elements. We have previously demonstrated the effectiveness of a FOXP3 cDNA in rescuing loss of FOXP3 via lentiviral vector delivery to patient-derived T cells in vitro and in vivo. In Aim 1 we will optimize the CRISPR/Cas9 system to d i s r u p t a n d restore FOXP3 gene expression in T cells, first in healthy donor, then in IPEX patient T cells to show reconstitution of Treg function. In Aim 2, we will restore FOXP3 gene expression in I P E X p a t i e n t CD34+ HSPCs. For both we will perform key determinations of target specificity by sequencing on and off-target sites in modified T-cells and CD34+ cells after transplantation into NSG/NSGDR1 mice. These studies will optimize CRISPR/Cas9 technology for efficient gene editing in primary cells. Our ultimate goal is to deliver autologous FOXP3 edited cells as a cure for IPEX syndrome, an otherwise deadly genetic autoimmune disease. In addition, we anticipate that these studies will be of broad relevance to other monogenic immunodeficiencies, especially those involving immune dysregulation.